An integrated approach to identify normal tissue expression of targets for antibody-drug conjugates: case study of TENB2

Abstract

Background and purpose: The success of antibody-drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target-mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen-independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance.

Experimental approach: A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by non-invasive single photon emission computed tomography - X-ray computed tomography imaging and immunohistochemistry.

Key results: The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target-mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As a proof of concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade.

Conclusions and implications: Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues.

Figure 1

Structural depiction of the unconjugated, unlabelled mAb used for pre-dosing (anti-TENB2, left) and the radiolabelled anti-TENB2-MMAE under evaluation ([¹¹¹In]-DOTA-anti-TENB2-MMAE, right). The auristatin drug, MMAE (green), was conjugated site-specifically to exactly two engineered thiols via the protease-labile maleimido-caproyl-valine-citrulline-para-amino-benzyloxy carbonyl (MC-vc-PAB) linker, while the radiometal chelate, DOTA (yellow), was randomly conjugated to lysine residues through amide bonds.

Figure 2

Mean (±SD) total antibody plasma concentration–time (by ELISA) profile of anti-TENB2-MMAE (ADC) at various doses in non-tumour-bearing SCID mice. PK data for the corresponding unconjugated anti-TENB2 mAb at 5 mg·kg⁻¹ are included for comparison.

Figure 3

Dose-dependent and antigen-specific blood concentration–time profile and tissue distribution of [¹¹¹In]-anti-TENB2-MMAE (ADC) in non-tumour-bearing SCID mice at 72 h after i.v. injection. The tracer was administered alone (red), in combination with unconjugated anti-TENB2 (ThioMab) at 1 (blue) or 10 (black) mg·kg⁻¹, or in combination with 10 mg·kg⁻¹ of an isotype-matched control antibody, anti-STEAP1 (green). (A) Whole blood PK data, plotted on a linear scale in a dose-normalized manner as % of injected dose mL^-1 of blood, derived by gamma counting in tissue distribution studies. (B) Tissue distribution of [¹¹¹In]-anti-TENB2-MMAE (ADC) in male SCID mice at 72 h expressed as mean %ID g⁻¹ ± SEM for three mice per group. Asterisks indicate statistical significance (P < 0.05) by one-way anova followed by Tukey post-test.

Figure 4

Comprehensive tissue distribution (A) and mass balance analysis (B) of [¹¹¹In]-anti-TENB2-MMAE (ADC) in non-tumour-bearing SCID mice at 72 h after i.v. injection. The tracer was administered alone or in combination with unconjugated anti-TENB2 (ThioMab) at 10 mg·kg⁻¹. Data are presented as % of injected dose g^-1 tissue (A) or % of injected dose (B) as mean ± SD for three mice per group. ‘Carc’ denotes carcass remaining after tissue harvest. Asterisks indicate statistical significance (P < 0.05) by unpaired t-test.

Figure 5

Radioactive anti-TENB2 was stable in plasma over the course of the study, and its catabolites were excreted in urine. No plasma protein complex formation was evident by HPLC analysis at 24 (red), 48 (blue) and 72 (green) h post-injection. Consistent presence of the radioimmunoconjugate [retention time (RT) ∼17 min, see Supporting Information Fig. S4] was observed in plasma of mice receiving [¹¹¹In]-anti-TENB2-MMAE (ADC) at tracer only (A) or 10 mg·kg⁻¹ (B) dose levels. In contrast, no intact protein and only radiolabelled catabolites (RT ∼24 min) were observed in urine from mice receiving tracer only (C) or 10 mg·kg⁻¹ (D) dose. Note the difference in y-axis scale, particularly between (C) and (D).

Figure 6

SPECT-CT fusion images of non-tumour-bearing SCID mice receiving [¹¹¹In]-anti-TENB2-MMAE (ADC) at tracer only (A, C, E) or 10 mg·kg⁻¹ (B, D, F) dose levels at 3, 48 and 72 h after i.v. injection (A–B, C–D and E–F respectively). Three-dimensional volume rendering images from sagittal (left) and coronal (centre) perspectives and coronal tomographic images (right) are shown in each panel. Note that the high signals above the thorax and below the animals' heads are both caused by reconstruction edge artefacts that are more severe at early time points when higher levels of radioactivity remain in the blood pool.

Figure 7

Representative immunohistochemical staining of TENB2 expression in the large intestine of a naïve, non-tumour-bearing SCID mouse. Large intestine exhibited moderate to strong staining by a TENB2-specific antibody (A) in a cytoplasmic distribution pattern. In contrast, a negative control antibody (B) did not result in staining of large intestine.

Figure 8

Comparative disposition of ¹²⁵I- and ¹¹¹In-labelled anti-TENB2-MMAE in the LuCaP 77 tumour explant model with and without a blocking dose of anti-TENB2. Dual-label tracers were co-administered at 0.1 mg·kg⁻¹. Blood concentration–time profiles of anti-TENB2-MMAE labelled with ¹²⁵I (A) and ¹¹¹In (B) were obtained in mice (n = 3) that received no block (red and green, respectively) or an i.v. anti-TENB2 blocking dose of 10 mg·kg⁻¹ (blue and black, respectively) 24 h before administration of the tracer. Tissue distribution (n = 3) of anti-TENB2-MMAE labelled with ¹²⁵I (C, E) and ¹¹¹In (D, F) in mice from the same four treatment groups was also performed at 24 (C, D) and 72 (E, F) h after radiotracer administration. *P < 0.05 by unpaired t-test.